Project Summary: The goal of our R01 titled ?Crosstalk between brassinosteroid and autophagy pathways in the regulation of plant growth and stress responses? is to determine how growth, development, and stress responses are coordinated in Arabidopsis, a model plant with extensive genetic, genomic and proteomic resources. To accomplish this we are carrying out detailed mechanistic studies to provide insights into fundamental biological processes, steroid hormone signaling and autophagy, that are conserved across eukaryotes. Specifically we are testing the hypothesize that brassinosteroid and autophagy pathways crosstalk through multiple mechanisms to coordinate plant growth and stress responses: (a) upon phosphorylation by BIN2, the autophagy receptor DSK2 acts as a phospho- regulated autophagy receptor for transcription factor BES1, and BES1 ubiquitination therefore leads to its degradation by selective autophagy. This in turn slows down plant growth under stress conditions; (b) brassinosteroids regulate TOR to promote growth and inhibit autophagy through BIN2 phosphorylation of TOR. These studies rely on nanoflow liquid chromatography coupled to mass spectrometry (LC-MS) based proteomics carried out in Co-PI Walley's lab to quantify ubiquitination and phosphorylation at the individual protein and proteome-wide levels. In this R01 revision, we are proposing to update our liquid chromatography for proteomics to enable 1) increased sample throughput and 2) improved chromatographic separation of peptides for greater protein and posttranslational modification identification. To achieve this we propose to purchase a Thermo Scientific Ultimate 3000 RSLCnano system, which is used extensively in the proteomics field.